Method of enhancing the effect of t-PA

ABSTRACT

The absorption rate of protein-thrombolytic agents with medicinal properties in the blood is enhanced by administering the protein intramuscularly together with an absorption enhancing agent, e.g. hydroxylamine or a salt thereof.

This application is a continuation-in-part of application 638,695, filedAug. 8, 1984, the entire disclosure of which is hereby incorporated byreference and relied upon.

This invention relates to the treatment of coronary prone individuals inthe throes of a suspected myocardial infarction in such a way as tominimize damage to the heart muscle and, more particularly, toimprovements in such treatments enabling the same to be commenced at theearliest possible time, even before direct qualified personal care ofthe individual can be established.

When a clot forms in a blood vessel, the body organ being supplied withblood by that blood vessel is compromised or totally deprived of bloodsupply. Depending on the blood vessel in which this occurs, the threatto the life of the individual is either small or very great as in thecircumstances to be addressed by the material below, i.e. certain lifethreatening circumstances. Clot formation in a vessel is described asthrombosis. Substances which dissolve thrombi are called thrombolyticsubstances. When a coronary artery clot is dissolved, the resultantestablishment of blood flow to the heart is called reperfusion.

Examples of life threatening or very serious clot formation in arterialvessels are cerebral thrombosis, renal thrombosis, opthalmic arterythrombosis, and very importantly, thrombosis of a coronary artery. Inapproximately 85% to 90% of cases of acute myocardial infarction(coronary heart attack), a thrombus is found in the coronary arterypreventing blood from flowing to the heart muscle (myocardium) andsupplying it with essential oxygen and other nutrients. A consequence ofa thrombus or clot forming in a coronary artery is the danger to themyocardium (heat muscle tissue that does the pumping of blood). Heartmuscle deprived of it's blood supply does not die immediately but doespromptly begin the process of becoming dead. The extent of the damagewhich is done to the heart muscle is, therefore, a function of the timeduring which the supply of blood to the infarct zone is restricted bythe occuluding thrombus.

Heretofore, the procedures undertaken to actually establish reperfusionto the infarct zone have generally been undertaken in a hospitalenvironment or equivalent. The so-called "prehospital38 treatment was,in general, directed toward keeping the patient alive and getting thepatient into the hospital environment as soon as possible so thattreatment minimizing the heart muscle damage could be accomplished.

The treatment undertaken in the hospital environment involves certainprocedures for establishing reperfusion in the infarct zone of thepatient's heart. When immediate surgery was not clearly indicated, theestablishment of reperfusion was accomplished by procedures which hadthe effect of unblocking the occlusion. The available proceduresincluded mechanical catheterization and the administration ofthrombolytic agents. Known thrombolytic agents, such as streptokinase orurokinase required intracoronary infusion or the slow infeed of theagent within the vessel at the site of occlusion by means of a catheter.In recent years, intravenous infusion of streptokinase has been shown tobe effective.

More recently a substance called tissue-type plasminogen activator ort-PA has been utilized experimentally. (The New England Journal ofMedicine, Mar. 8, 1984, Volume 310, No. 10, pages 609-613). Unlike otherplasminogen activators, such as streptokinas or urokinase, t-PA--whichis found in only small amounts in the body--acts specifically on clotsand not on other relevant proteins in the blood, when maintained atappropriate and effective levels.

A 1984 Commentary found in Biochemical Pharmacology, Volume 33, No. 12,pages 1831-1838 entitled

"Coronary Thrombolysis: Pharmacological Considerations With Emphasis OnTissue-Type Plasminogen Activator (t-PA)" contains the followingconclusionary statement:

"Selection of pharmacological agents for induction of coronarythrombolysis has been determined largely by availability. Unfortunately,both streptokinase and urokinase induce a systemic lytic state withdepletion of circulating fibrinogen, plasminogen, and α 2-antiplasmin,and accumulation of fibrin degradation products. All of these factorsconspire to set the stage for hemorrhage with a risk of seriousbleeding. Intravenous administration of these agents is limited by alower success rate, in part because the upper bound of dose isconstrained by the risk of inducing a severe systemic lytic state.

The probability that progress in recombinant DNA technology will lead towidespread availability of tissue-type plasminogen activiator isparticularly exciting because of the clot specific properties of t-PA.For coronary thrombolysis its potential advantages include: safety andefficacy of intravenous administration of high doses; effective clotlysis without induction of a systemic lytic state; prompt implementationwithout the need for extensive characterization of the coagulation andfibrinolytic systems in each patient prior to and during therapy;avoidance of frank allergic reactions or variations in dose-responserelation due to immune complex formation; ease of minute-by-minuteadjustment of dosage and prompt termination of fibrinolysis when neededbecause of the short biological half-life of t-PA and the lack ofinduction of a systemic lytic state".

The promise attributable to t-PA administration was discussed at a newsconference at a meeting of the Americal Heart Association and reportedby the New York Times on Nov. 16, 1983, in an article entitled, "ProteinOf Cancer Cells Used To Halt Coronaries." The article refers toinjection of t-PA by stating the following: "The protein t-PA can simplybe injected into the vein in the arm of the patient seized by amyocardial infarction or heart attack, and it travels through the bloodto dissolve a clot, in much the same way as Draino clears up stoppedplumbing."

The article further indicated under the subheading "Hopes For FutureApplication" that many physicians have expressed excitement aboutresearch into the use of t-PA to treat heart attacks because they hopethat some day it may be used in emergency rooms and ambulances to stopheart attacks at their earliest stages before they kill or causepermanent damage. Under the "Hopes For Future Application" subheadingthere is also included the following paragraph: "Dr. Burton E. Sobel OfWashington University, one of the researchers, speculated that patientsmight some day carry a vial with them so that the drug could be injectedimmediately after they felt chest pain and other early symptoms of aheart attack."

In medical parlance, a vial is a container for a quantity of liquidmedicine or diluent having a rubber stopper capable of being pierced bya hypodermic needle of a syringe to enable the operator of the syringeto withdraw a predetermined dosage of the liquid from the vial. In thecase of t-PA as currently used, the dosage could then be injected intothe mother liquid container of an infusion assembly. The necessity toadminister the drug by intravenous infusion or by intravenous injectionpresents a significant barrier to self-administration from a practicalview point, particularly when considering the disconcertingcircumstances of the individual undergoing the symptoms of a myocardialinfarction.

The development of an effective self-administration procedure for t-PAsufficient to enable its utilization by a targeted coronary proneindividual immediately following onset of symptoms, would materiallyincrease the potential efficacy of t-PA as a thromobolytic agent byinsuring its use at the earliest possible time often before irreversibleheart muscle damage has occurred, and, at the same time, provide atreatment of the pre-hospital or pre-ambulance type which for the firsttime is directly effective to minimize heart muscle damage accompanyingmyocardial infarction. It is an object of the present invention toprovide such a self administering treatment.

Another object is to enhance the absorption rate of t-PA in the bloodwhen introduced intramuscularly.

The invention includes packaging t-PA and an agent enhancing theabsorption of t-PA in the blood. The agent preferably is hydroxylaminehydrochloride, in a known emergency type automatic injector andinjecting the two medicament agents into the muscle tissue, e.g. afterhaving received a decision to do so over the telephone from a qualifiedsource and at a time prior to the establishment of direct contactqualified personal care.

Even though t-PA may be regarded as a clot selective thrombolytic agent,when introduced into the blood stream at a predetermined level, teststhus far performed show that the concentration can be increased to thepoint that a systemic lytic state can be induced. Intramuscularinjection involves the introduction of concentrated dosage of t-PA in anarea contiguous to and substantially surrounding the wound caused by thepenetration and withdrawal of the injection of the hypodermic needle.Consequently, it would be expected that at least a localized lytic statewould be induced resulting in hemorrhage from the needle wound.Unexpectedly, tests have shown that no such hemorrhage does in factoccur.

Second, t-PA is a large protein. It would not be expected that it wouldbe absorbed into the blood stream in discernible quantities.Extravascular levels of protein are about 1/10 that of intra-vascularprotein. It is thought that this is so because the capillary poresthrough which transport of protein can occur are small relative to themolecular size of protein and limit protein transport because ofelectrical charge. It was thus highly problematical as to whether alarge protein such as t-PA, when given intra-muscularly, i.e. outsidethe blood vessels, would find its way rapidly into the blood stream indiscernible quantities. Application tests have indeed shown that byitself t-PA does not find its way rapidly into the blood stream intherapeutically significant quantities after intramuscular injection.

The actual treatment of the system must therefore include intramuscularinjection of an absorption enhancing agent simultaneously orsubstantially simultaneously with the intramuscular injection of thet-PA so as to produce effective thrombolytic blood levels.

Augmentation of absorption of low molecular weight substancesadministered topically, subcutaneously, or intramuscularly has beenachieved with vehicles such as dimethylsulfoxide (DMSO) and byenhancement of skeletal muscle blood and lymph flow.

However, DMSO has proven ineffective as an absorption enhancing agentfor t-PA.

In accordance with the principles of the present invention, theabsorption rate of t-PA in the blood is enhanced by utilizing with thet-PA dosage, a dosage of an absorption enhancing agent for t-PA,preferably hydroxylamine hydrochloride. Preferably, the absorptionenhancing agent such as hydroxylamine hydrochloride is mixed in with thet-PA dosage to form a single mixed dosage which is then injectedintramuscularly (i.m.), e.g. as described in the parent application.Through the contemplation of the present invention to inject theabsorption enhancing agent as a separate dosage within the same site asthe separate dosage of t-PA, (e.g. U.S. Pat. No. 4,394,863). An exampleof an amount of absorption enhancing agent, such as hydroxylaminehydrochloride, which is added to the t-PA dosage, as previouslydescribed, to form a single mixed dosage is an amount of from 0.1 to 85e.g. 0.1 to 40 or 1 to 85 milligrams per kilogram of body weight. As theabsorption enhancing agent hydroxylamine is preferably employed in theform of a non-toxic water soluble salt. Thus there can be used forexample in place of hydroxylamine salts such as hydroxylaminehydrochloride, hydroxylamine hyrobromide, hydroxylamine hydroiodide,hydroxylamine sulfate, hydroxylamine nitrate, hydroxylamine acetate, andhydroxylamine propionate. Most preferably there is employedhydroxylamine hydrochloride.

There is also contemplated as absorption enhancing agents for t-PA inaccordance with the invention compounds such as ammonia (ammoniumhydroxide), ammonium carbonate and other ammonium salts, e.g. ammoniumchloride, ammonium acetate, ammonium bromide and ammonium sulfate, urea,mono and dialkyl ureas, e.g. methyl urea, ethyl urea, propyl urea, butylurea, N,N-dimethyl urea, N,N-diethyl urea, N,N-diisopropyl urea, monoand diaryl ureas, e.g. phenyl urea, p-tolylurea, N,N-diphenyl urea and,N,N-di-p-tolyl urea, thiourea, hydantoin, 5-substituted hydantoins, e.g.5-alkyl, 5-aralkyl, and 5-aryl hydantoins and 5,5-dialkyl and 5,5-diarylhydantoins, e.g. 5-methyl hydantoin, 5-ethyl hydantoin, 5,5-dimethylhydantoin, 1,5-trimethylene hydantoin, 1,5-tetramethylene hydantoin,5-phenyl hydantoin, 5-p-tolyl-hydantoin, and 5,5-diphenyl hydantoin,guanidine, methyl guanidine, hydrazine, alkyl and aryl hydrazines, e.g.methyl hydrazine, ethyl hydrazine, butyl hydrazine, phenyl hydrazine anddiphenyl hydrazine, alkyl and aryl hydroxylamines, e.g. methylhydroxylamine, ethyl hydroxylamine and phenyl hydroxylamine. Thesubstituted ureas, hydrazines and hydroxylamines likewise can be used inthe form of salts, e.g. as hydrochlorides.

Also while the simultaneous administration of t-PA and absorptionenhancing agent is primarily intended for human use, it is within thescope of the invention that they be administered to other mammals, e.g.dogs, cats, cattle, and horses.

It is known that hydroxylamine, e.g. as the hydrochloride, dissociatest-PA from its naturally occurring inhibitor in tissue culture, Levin.Proc. Natl. Acad. Sci. USA 80, 6804-6808 (1983). It is also known thathydroxylamine inhibits platelet aggregation, see Iizuka, Chem.Pharmacol. Bull. 20 614-616 (1972) and elicits smooth muscle relaxationpotentially enhancing vasodilation and hence absorption at the injectionsite, see Diamond, J. Pharmacol, Exp. Therap. 225, 422-426 (1983). Theseproperties may contribute to its success in the present invention.

While t-PA and the absorption enhancing agent would usually beadministered intramuscularly they can also be administered singly or incombination intravenously since hydroxylamine has been shown (see Levin,loc. cit) to disassociate t-PA inhibitor from t-PA, thereby enhancingthe effect of the infused exogenous t-PA or the hydroxyl amine and thusreducing the amount of t-PA required to accomplish thrombolysis. Aspointed out above the hydroxylamine will usually be administered in theform of a non-toxic salt, preferably the hydrochloride. The dosage ofabsorption enhancing agent, e.g. hydroxylamine hydrochloride can be inthe range previously mentioned. In place of hydroxylamine in this placeof the invention there can be added other t-PA inhibitor disassociatingagents. In accordance with the teachings of my copending U.S.application 460,011, filed Jan. 21, 1983 (the disclosure of which ishereby incorporated by reference into the present specification),electrical stimulation of the muscle at the injection site was employedin concert with the inclusion of an absorption-enhancing agent,specifically hydroxylamine hydrochloride, in the injectate in a numberof the following examples using intramuscular injection. Electricalstimulation augments and enhances the absorption of the absorptionenhancing agent of the invention.

Although as pointed out in the parent application an automatic injectordevice suitable for intramuscular self-administration of t-PA can beemployed, the examples set forth below were performed by administeringthe t-PA and hydroxylamine hydrochloride directly into the muscle with aconventional needle and syringe. Administration of the agent with anautomatic injector, however, it is believed will lead to even higherblood levels than those obtainable by manual injection.

After an approach employing intramuscular injection of t-PA withhydroxylamine (as the hydrochloride) and electrical stimulation ofskeletal muscle at the injection site in rabbits had been found to yieldpeak blood levels of t-PA comparable to or exceeding those known toelicit coronary thrombolysis after intravenous infusion of t-PA in dogsand in patients, an analogous approach was evaluated in dogs subjectedto coronary thrombosis. Facilitated absorption of t-PA afterintramuscular injection was found to elicit coronary thrombolysis aswell as therapeutic blood levels of t-PA in these feasibilityexperiments.

Large injectate volumes were employed because of the limited solubilityof t-PA in conventional buffers. For consistency the volumes used inrabbits were selected to be similar to those planned for use in dogs (1and 1.5 ml per injection site for rabbits and dogs respectively) eventhough they represented large volumes with respect to rabbit musclemass. Thus the same concentration of absorption-enhancing agent per mlof injectate was used in both species even though they resulted inadministration of markedly greater amounts of hydroxylamine per kg ofbody weight and a 10-fold lower concentration of t-PA in the injectatesin rabbits compared with dogs despite administration of comparableproportion of t-PA administered per kg of body weight in the twospecies. Concentrating the t-PA appreciably with solubilizing agentssuch as thiocyanate it is believed will permit the volumes to be reducedsubstantially.

For studies in rabbits, the t-PA employed was either harvested frommelanoma cell supernatant fractions (mt-Pa) as previously described(Bergmann, Science 220 1181-1183 (1983) or produced by recombinant DNAtechnology, Van der Werf, Circulation 69 605-610 (1984) (rt-PA,Genentech Corp., lot BH004 DAX). Results with the two preparations wereindistinguishable and therefore the preparations were pooled.Concentrations of 0.5 mg t-PA per ml buffer (0.3 M NaCl, 0.01% Tween 80,0.01M potassium phosphate buffer pH 7.5) were used. For studies in dogs,rt-PA (Genentech, lot TE031A) was concentrated 20-fold with an Amiconmembrane filter system.

DMSO was used in 1% or 3% (v/v) solutions in vitro and in injectates.Hydroxylamine hydrochloride was used in concentrations of 43.75 mg perml of t-PA solution. This concentration was compatible with a totalhydroxylamine hydrochloride dose of approximately 13 mg/kg shown to bewell tolerated physiologically.

To determine the extent to which the absorption-enhancing agentsevaluated might interact with t-PA, solutions of rt-PA (0.015 to 50ng/ml) were incubated at 37° C. for 1 hour after addition of 1% DMSO, 3%DMSO, 175 mg/ml hydroxylamine (as the hydrochloride), or both DMSO andhydroxylamine (as the hydrochloride). No effects were discernible ont-PA assayed innumoradiometrically or functionally.

Studies were performed in 56 nonfasted, white male New Zealand rabbitsweighing approximately 2 kg. Endogenous t-PA in these animals does notreact with antibody prepared against human t-PA and hence does notinterfere with the immunoradiometric assay used to characterize bloodlevels of exogenously administered t-PA. Animals were anesthetized withsodium pentobarbital (24 mg/kg) and ventilated with 95% oxygenadministered through a tracheostomy at 2l/min. Skeletal muscle (vastusmedialis) at the injection site was exposed bilaterally and serial bloodsamples were drawn through an indwelling femoral venous catheter. Toaugment skeletal muscle blood and lymph flow at the injection site, themuscle was stimulated for 2.0 msec at 14 volts with five pulses persecond with two 27-gauge, 0.5 inch stainless steel needles. A singlenegative distal electrode was used as well. A total of 1 mg of t-PA/kgbody weight was injected manually divided in 1 ml aliquots in each of 4sites.

Coronary thrombosis was induced in fasted anesthetized dogs weighingapproximately 23 kg, see Bergmann Science 220. 1181-1183 (1983).Occlusive thrombus formed within five to 10 minutes and was confirmedangiographically. Serial venous blood samples were obtained through anindwelling inferior vena caval catheter. Electrical field stimulation atthe injection site was implemented with three 27-gauge stainless steel,one serving as the negative reference. Parameters were the same as thoseused in rabbits. t-PA was injected directly into exposed sartoriusmuscle in 1.5 ml aliquots per site such that the total dose was 3 mg/kgbody weight and the total volume of injectate was 6 ml in aggregate foreach dog.

The primary endpoint for experiments in the 56 rabbits studies was t-PAactivity in blood. t-PA antigen levels were assayed serially aspreviously described Bergman, loc. cit. and Van der Werf, No. Engl. 2Med. 310, 609-613 (1984). Functional t-PA activity was determined aswell Bergman, loc. cit and Tiefenbrunn, Circulation 71, 110-116 (1985).Blood samples were obtained at 0° to 4° C. in sodium citrate vacutainertubes before intramuscular injection of t-PA or vehicle alone,immediately after injection, and at selected intervals from one to 60minutes subsequently.

For the feasilibity experiments in dogs, an additional endpoint wascoronary thrombolysis documented angiographically. Blood pressure, heartrate, the electrocardiogram, arterial blood gases and pH, hemoglobin andhemoglobin oxygen saturation were monitored.

For experiments in both species, a crude assessment of potential muscleinjury at the site of injections was made by gross inspection. Inaddition, serial blood samples were assayed for plasma creatine kinase(CK) activity spectrophotometrically, Klein, Cardiovasc. Res. 7, 412-418(1973) in view of the known prompt and marked liberation of CK into thecirculation when skeletal muscle is inured.

Serial changes in blood levels of t-PA were evaluated in 56 rabbitscomprising several groups. Blood levels were assessed before and atselected intervals after intramuscular injection of buffer with orwithout absorption-enhancing agent alone; or t-PA in buffer, buffer withDMSO, buffer with hydroxylamine (as the hydrochloride), or buffer withDMSO and hydroxylamine (as the hydrochloride).

The same combinations were evaluated with and without concomittantelectrical stimulation of muscle at the injection site throughout theblood sampling interval. Once it had been determined that hydroxylaminefacilitated absorption of t-PA, experiments were performed to define thedose-response relations for absorption of t-PA as a function to theconcentrations of t-PA and the concentration of hydroxylamine in theinjectate. Possible systemic effects of hydroxylamine on absorption oft-PA wer assessed in rabbits by administering hydroxylamine without t-PAin two injection sites and t-PA without hydroxylamine in the other twosites.

The experiments performed in dogs were undertaken after it had beendetermined with rabbits therapeutic blood levels could be induced withamounts of t-PA/kg body weight (1 mg/kg) of the same order of magnitudeas those that had been used previously for intravenous administration oft-PA in patients (0.5 to 0.75 mg/kg). Intramuscular t-PA wasadministered with hydroxylamine (as the hydrochloride) within five to 45minutes after angiographic documentation of formation of an occlusiveclot in the left anterior descending coronary artery, generallyoccurring within seven to 10 minutes after introduction of thethrombogenic coil into the vessel. Serial aniography was performed atapproximately 15 minute intervals. Effects of t-PA on coronary thrombicorrelated with plasma t-PA levels. After clot lysis (approximately 15minutes after injection of t-PA), heparin (500 U/kg body weight) wasgiven to prevent reocclusion. In the absence of exogenous activation ofthe fibrinolytic system, clots induced by the indwelling thrombogeniccoronary arterial coil invariably persist despite administration ofheparin (n=40 dogs). Statistical comparisons were performed by analysisof variance with Bonferroni critical limits or with Students test forpaired data. Values are expressed as means ±SE.

Effects of Absorption-Enhancing Media on t-PA Activity in vitro

Neither hydroxylamine (as the hydrochloride) (175 mg/ml), 1% DMSO, 3%DMSO, nor concomitant hydroxylamine (as the hydrochloride) and DMSOmodified immunoradiometrically detectable t-PA or functionallydetectable t-PA activity in samples incubated for 1 hour at 37° C.containing 0.015 to 50 ng rt-PA.

Concentrations of t-PA in Blood

Prior to intramuscular injection of rt-PA, no human t-PA was detectableby immunoradiometric assay in plasma from any of the rabbits. Nodetectable endogenous t-PA activity was evident in plasma samplesassayed with the fibrin plate functional assay despite the minorsurgical procedure performed and the imposed electrical stimulation ofmuscle for 60 minutes in any of four rabbits tested. No human t-PA wasdetectable after injection of any of the combinations of vehicles testedwhen exogenous t-PA was not included in the injectate. Noimmunoradiometrically detectable t-PA was present in plasma samples fromsham operated dogs during a 60 minute sampling interval with or withoutintramuscular injection of a total of 262 mg/ml of hydroxylamine as thehydrochloride administered in multiple sites. Fibrin plate assayablefunctional activity in sham operated dogs ranged from 10 to 53 IU/ml anddid not increase in any of four animals tested during the 60 minutesampling interval after electrical stimulation and intramuscularinjection of hydroxylamine hydrochloride in buffer without t-PA.

In control experiments with hydroxylamine hydrochloride alone (262 mg)injected intramuscularly in dogs, peak methemoglobin levels ranged from11 to 13% and occurred within five to 15 minutes after intramuscularinjection (n=3). Arterial oxygen tension decreased to a minimum of 93 mmHg. Hemoglobin saturation with oxygen declined to a minimum of 81%.Except for transitory acceleration of heart rate, dogs givenhydroxylamine hydrochloride with or without t-PA exhibited nosignificant hemodynamic or electrocardiographic abnormalities.

IN THE DRAWINGS

FIG. 1 is a graph of immunoradiometrically detectable and functionallyactive plasma t-PA activity in plasma samples from a rabbit injectedwith 2 mg t-PA buffer with 43.75 mg/ml hydroxylamine hydrochloride(total injectate volume=4 ml divided among 4 sites) followed byelectrical stimulation at the injection sites throughout the samplinginterval. Both immuno-reactive and functionally active t-PA peakedrapidly after intramuscular injection with facilitated absorption.

FIG. 2 is a graph showing the dependence of the peak concentrationplasma of immunoradiometrically detectable t-PA on the concentration ofhydroxylamine in the injectate. Conditions were the same as thoseindicated in the legend to FIG. 1 except that the amounts ofhydroxylamine hydrochloride in the 4 ml aggregate volume of injectatewere varied as indicated in the figure.

FIG. 3 is a chart showing peak plasma t-PA activity as a function of theamount of t-PA administered intramuscularly in 6 rabbits. Conditionswere the same as those indicated in the legend to FIG. 1 except that thetotal amount of t-PA administered was varied as indicated. Panel Adepicts immunoradiometrically detectable activity; panel B depictsamidolytic, functional activity. Dose related differences throughout the1 hour interval of measurement for the entire time-activity aerol (n=30determinations) were significant as determined byanalysis of variance(p<0.001).

FIG. 4 is a graph showing early changes in plasma t-PA concentrationsafter facilitated absorption of intramuscularly administered t-PA ineach of three rabbits. Conditions were the same as those indicated inthe legend to FIG. 1.

FIG. 5 a graph of serial changes in plasma t-PA assayedimmunoradiometrically in a dog which had been subjected to coronarythrombosis. Thrombosis was induced with a thrombogenic coil advancedinto the left anterior descending coronary artery at the tip of acoronary arterial catheter. Coronary thrombolysis was induced byfacilitated absorption of intramuscularly administered t-PA. (Thethrombogenic coil elicited formation of a clot evident by lack of distalfill with angiographic dye as well as by lack of opacification of thevessel proximal to the coil that appears as a bright rectangle). Fifteenminutes after intramuscular administration of t-PA (3 mg/kg in a totalinjectate volume of 6 ml divided among four sites) and electricalstimulation of muscle at the injection site, lysis of the clot proximaland distal to the coil was evident with angiographically demonstrablerestoration of patency. As can be seen, plasma t-PA activity peaked soonafter facilitated absorption of intramuscularly administered t-PA.Elevated levels persisted throughout the sampling interval. A secondarypeak was seen in each of the three dogs studied.

Blood Levels of t-PA After Intramuscular Injection In Rabbits

As shown in Table 1, t-PA injected in buffer alone increased bloodlevels only minutely. The addition of DMSO to the injectate did notincrease t-PA levels in plasma. In contrast, hydroxylamine hydrochlorideaugmented absorption of t-PA yielding peak blood levels five minutesafter injection approximately 40-fold higher than those seen in itsabsence. An example of serial changes of immunoradiometrically andfunctional t-PA activity assayed with fibrin plates after intramuscularabsorption of t-PA facilitated by inclusion of hydroxylaminehydrochloride in the injectate and electrical stimulation of muscle atthe injection site is shown in FIG. 1.

                  TABLE 1                                                         ______________________________________                                        Immunoradiometrically Detectable t-PA In Plasma (ng/ml)                       After Intramuscularly Administered t-PA                                                                        t-PA in                                                            t-PA in    Buffer +                                     Interval After                                                                           t-PA In    Buffer + 3%                                                                              Hydroxylamine                                Injection  Buffer Alone                                                                             DMSO       Hydrochloride                                (min)      (n = 6)    (n = 6)    (n = 15)                                     ______________________________________                                        0          0 ± 0   0 ± 0   0 ± 0                                     5          8 ± 2   11 ± 4  431 ± 52*                                 15         9 ± 2   8 ± 2   146 ± 16*                                 30         9 ± 2   9 ± 1    85 ± 17*                                 60         10 ± 3  10 ± 1   53 ± 11*                                 ______________________________________                                         Values are means ± SE. All injectates contained 2 mg tPA in an             aggregate of 4 ml (1 ml per site). The concentration of hydroxylamine         hydrochloride was 43.75 mg/ml. All experiments tabulated were performed       with electrical stimulation of muscle at the infarction site.                 *P < .01 compared with tPA in buffer alone or in buffer + DMSO.          

To determine whether augmentation of muscle blood flow by electricalstimulation would enhance absorption of t-PA administeredintramuscularly, experiments were performed with and without electricalstimulation after injection of t-PA in buffer alone, t-PA in buffersupplemented with DMSO, and t-PA in buffer supplemented withhydroxylamine hydrochloride. The very low blood levels seen when t-PAwas administered without hydroxylamine hydrochloride were notconsistently modified by electrical stimulation (n=11 animals). However,in animals given t-PA with hydroxylamine hydrochloride (n=15)stimulation augmented peak levels by an average of 258±32% withoutaltering the time course of absorption or clearance of t-PA.

As shown in FIG. 2, immunoradiometrically detectable t-PA peak bloodlevels were proportional to the amount of hydroxylamine hydrochloride inthe injectate. Addition of 1% or 3% DMSO to hydroxylamine (as thehydrochloride)-enriched injectates did not increase peak blood levels oft-PA compared with results with hydroxylamine hydrochloride alone whenthe amount of t-PA was held constant. Both immunoradiometricallydetectable and functionally active t-PA after administration ofexogenous t-PA were proportional to the concentration of t-PA over afour-fold range when the amount and concentration of hydroxylaminehydrochloride in the injectate were held constant (FIG. 3). As can beseen in FIG. 4, blood levels rose rapidly and peaked between 4 and 5minutes after injection. Appreciable concentrations of t-PA in plasmawere evident as early as one minute after intramuscular injection ineach case.

The augmentation of peak plasma t-PA after facilitated absorption withhydroxylamine hydrochloride was not caused simply by the decreased pH ofthe injectate. In each of two animals, the pH of the injectate wastitrated to 5.9 without hydroxylamine. Plasma t-PA concentration fiveminutes after injection was only 6 ng/ml. No significant increaseoccurred subsequently. The increment seen with hydroxylaminehydrochloride was not attributable simply to systemic effects ofhydroxylamine hydrochloride. In two animals in which hydroxylamine wasinjected into the right and t-PA in buffer into the left thigh muscle,peak blood levels did not exceed those in Table 1 for t-PA injected inbuffer alone.

Although the amounts of absorptionenhancing agent per kg body weightused in rabbits were considerably greater than those used in dogs oranticipated ultimately for possible clinical studies, the excessivelylarge quantities were employed to determine whether high concentrationsin the injectate would be deleterious to skeletal muscle. In rabbits,plasma CK was not significantly different 30 minutes after the surgicalprocedure, injection of t-PA with hydroxylamine hydrochloride andelectrical stimulation compared with values after injection of bufferalone under the same conditions (690±82 compared with 696±63 IU/l). Indogs given 175 mg hydroxylamine hydrochloride with or without t-PA,plasma CK increased by less than 18% of baseline at the completion ofthe study. No hematoma were evident by gross inspection. Lightmicroscopy of sections from the injection site obtained two hours afterinjection delineated only scanty interstitial hemorrhage andinflammation.

Effects of Facilitated Absorption of Intramuscularly Administered t-PAon Coronary Thrombolysis in Dogs.

After demonstrating that facilitated absorption of t-PA could beachieved in rabbits with hydroxylamine hydrochloride in the injectate,pilot studies were performed in dogs to determine whether the approachdeveloped could elicit coronary thrombolysis. Arterial blood pressureafter injection of hydroxylamine hydrochloride intramuscularly with(n=3) or without (n=3) t-PA declined only modestly (from an average of166/121 mm Hg to 144/104) reaching a minimum 2 minutes after injection.Heart rate increased transiently by an average of 32% peaking also 2minutes after injection. Ventricular arrhythmias did not occur withhydroxylamine hydrochloride alone. Intramuscularly administered t-PA (3mg/kg) followed by electrical stimulation initiated coronarythrombolysis within 15 minutes heralded by reperfusion arrhythmias.Similar results were obtained in each of the three animals studied.Plasma t-PA values followed a similar time course but were lower thanthose seen in rabbits. The differences may reflect species differencesin the absorption or clearance of human rt-OA or the larger ratio ofinjectate volume to muscle mass in rabbits. In addition, as shown inFIG. 5, a secondary peak of immunoradiometrically detectable t-PAoccurred beginning approximately 40 minutes after the first peak in eachdog compatible with lat release from the skeletal muscle depot becauseof changes in blood flow or slow lymphatic transport of t-PA into thecirculation among other possibilities.

Thus it has been found that therapeutic blood levels of functionallyactive t-PA can be achieved and that coronary thrombolysis can beeliicted by facilitated absorption of intramuscularly injected material.Plasma activity peaked within five minutes after injection andsubsequently declined rapidly, consistent with the known half-life oft-PA in the circulation. The blood levels obtained were sufficient toinduce coronary thrombolysis in dogs within 15 minutes despite thecontinued presence of an indwelling, coronary, thrombogenic coil.Absorption of t-PA was enhanced by inclusion of hydroxylamine in theinjectate and by augmentation of skeletal muscle blood flow byelectrical stimulation. Gross injury to skeletal muscle did not occur.

Because low levels of t-PA in plasma may be adequate to induce clotlysis of nascent thrombi judging from results of studies in vitro andbecause the biological half-life of t-PA bound to fibrin issubstantially longer than the half-life of circulating t-PA, seeBrommer, Thromb. Res. 34, 109-115 (1984), Tran-Thang, Blood 63 1331-1337(1984), Bergmann, Circulation 70 II:108 (Abstract) (1984), it isbelieved that coronary thrombolysis early after the onset of thrombosisin vivo may be obtained with lower quantities of t-PA, hydroxylaminehydrochloride, or both than those used in the examples set forth above.Reduction of the injectate volume would diminish the dose ofhydroxylamine or other absorption enhancing agent required and minimizepotential injury to muscle at the injection site.

To date, t-PA and other activators of the fibrinolytic system have beengiven only by direct injection into the blood stream. This inventionprovides an alternative means of administration of t-PA potentiallyamendable to prompt implementation by paramedical personnel or bytelephonically supervised patients at high risk previously instructed inself-medication procedures.

Hydroxylamine was employed after numerous attempts with otherabsorption-enhancing media for other compounds failed to yield thedesired results with t-PA. Its major side effect, induction ofmethemoglobinemia does not prohibitively limit tissue oxygenation withthe doses used. If the concentration of the hydroxylamine in theinjectate is the critical determinant of absorption of t-PA as appearslikely judging from the present results, the total dose of hydroxylaminerequired in human subjects is likely to be so low that inducedmethemoglobinemia would be of only trivial extent even for patients withischemic heart disease especially if the injectate volume can be reducedfurther by increasing the concentration of t-PA. In those cases wherethe methemoglobinemia accompanying use of this absorption-enhancer isdeemed to be unacceptably severe, adjuvant measures such as concomitantadministration of methylene blue or glutathione might be utilized tominimize or obviate the problem, see Layne, J. Pharmacol. Exp. Therap.165, 36-44 (1969).

Blood levels of t-PA comparable to those obtained in the presentinvestigation induce coronary thrombolysis in experimental animals andpatients without inducing a systemic lytic state predisposing tobleeding. The time course of elevation of plasma t-PA after facilitatedintramuscular absorption is particularly favorable because of its sharppeak. With the envisioned application of an appropriate regimen,subjects would be under direct medical care soon after self-medicationwith an automatic injector or treatment by relatives of paramedicalpersonnel. Thus, as the blood levels declined promptly afterintramuscularly administered t-PA had been given, intravenous infusionscould be initiated along with anticoagulants or other measures taken toprevent reocclusion while definitive diagnostic information wa beingobtained.

The possibility that myocardial reperfusion induced by facilitatedabsorption of intramuscularly administered t-PA might give rise toreperfusion arrhythmias is easily managed in the setting of the cardiaccatheterization laboratory or coronary care unit but can be potentiallydangerous in the medically unattended patient. Thus, there is advantagein the concomitant administration of an antifibrillatory oranti-arrhythmic agent such as lidocaine or an alpha-adrenergic blockingagent as set forth in the parent application.

It has also been found that to prevent reocclusions or plateletaggregation it is desirable to either:

1. inhibit synthesis of thromboxane A *thromboxane A₂) with athromboxane synthetase inhibitor, e.g. an imidazole such as4-(2-[1H-imidazol-1-yl]ethoxy)-benzoic acid hydrochloride (dazoxiben)

2. introduce an antagonist for the receptor of the thromboxane A(thromboxane A₂) such as [1α,2β(5Z), 3β(1E),4α]-7-[3-(3-cyclohexyl-3-hydroxy-1-propenyl)-7-oxabicyclo[2.2.1]hept-2-yl]-5-heptenoicacid) (SQ 27,427).

3. introduce another inhibitor of platelet aggregation, e.g. aspirin,indomethacin, naproxin, and sulfinpyrazone.

The agent for the prevention of reocclusions or platelet aggregationscould be administered simultaneously or sequentially in either orderwith reference to the t-PA and absorption enhancing agent, e.g.hydroxylamine hydrochloride. The agent for the prevention ofreocclusions or platelet aggregations can be administered inconventional manner, e.g. intramuscularly, intravenously, or evenorally.

The receptor antagonist or other agent for prevention of plateletreocclusions can be administered for example in an amount of 0.1-10mg/kg body weight.

What is claimed is:
 1. A method of increasing the .[.absorption.]..Iadd.effect .Iaddend.of t-PA in the blood in a mammal in need of t-Patherapy comprising administering to the mammal .Iadd.exogenous t-PA and.Iaddend.an amount of t-PA inhibitor disassociating agent effective toincrease the disassociation of the inhibitor from the t-PA, the amountof exogenous t-PA being less than that required to be administered inthe absence of the inhibitor disassociating agent.
 2. A method accordingto claim 1 wherein the inhibitor dissociating agent is addedintramuscularly.
 3. A method according to claim 1 wherein the inhibitordisassociating agent is added intravenously.
 4. A method according toclaim 1 wherein the inhibitor disassociating agent is hydroxylamine or anon-toxic salt thereof.
 5. A method according to claim 4 wherein theinhibitor dissociating agent is added intramuscularly.
 6. A methodaccording to claim 4 wherein the inhibitor disassociating agent is addedintravenously.
 7. A method according to claim 4 wherein the inhibitordisassociating agent is hydroxylamine hydrochloride.